Substituted 7- and/or 9-amino tetracyclines



United States Patent 3,345,410 SUBSTITUTED 7- AND/0R 9-AMINO TETRACYCLINES Robert Winterbottom, New City, Panayota Bitha, New York, and Henry Marcel Kissman, Nanuet, N.Y., as signors to American Cyanamid Company, Stamford, Conn., a corporation of Maine No Drawing. Filed Dec. 1, 1966, Ser. No. 598,206 10 Claims. (Cl. 260--559) ABSTRACT OF THE DISCLOSURE This disclosure describes compounds of the class of 7- and/or 9-mono(lower alkyl)aminoor -di(lower alkyl) aminotetracyclines useful as antibacterial agents.

This application is a continuation-in-part of our 00- pend-ing application Ser. No. 533,820, filed Mar. 14, 1966, now abandoned.

This invention relates to new compounds of the tetra cycline family and, more particularly, is concerned with novel substituted 7 and/or 9-amino tetracyclines which may be represented by the following general formula:

wherein R is hydrogen or hydroxy, R is hydrogen or methyl, and R and R are hydrogen, mono(lower alkyl) amino or di(lower alkyl)arnino with the proviso that R, and R cannot both be hydrogen. Typical compounds represented by the above general formula are, for example,

7-methylamino-6-demethyltetracycline, 7-diethylamino-6-demethyltetracycline, 7-isopropylamino-o-demethyltetracycline, 9-ethylarnino-6-demethyltetracycline, 9-isopropylamino-6-demethyltet-racycline,

7,9-di (ethylamino) -6-demethyltetracycline, 7-dimethylamino-6-demethyltetracycline, 9-methylamino-G-demethyltetracycline,

7 -methylamino-9-ethyl-arnino-6-demethyltetracycline, 7-methylaminotetracycline,

9-ethylaminotetracycline,

7 -isopropylamino-5 -hydroxytetracycline, 9-methylarnino-S-hydroxytetracycline, 7-ethylamino-5-hydroxy-6-demethy1tetracycline, and 7 ,9-di methylamino) -S-hydroxy-o-demethyltetracycline.

These new tetracycline derivatives may be prepared by a reductive alkylation process comprising interacting a compound of the following general formula:

wherein R and R are as hereinabove defined and Y and Z are hydrogen, nitro or amino with the proviso that Y Patented Oct. 3, 1967 and Z cannot both be hydrogen, with a carbonyl compound of the following general formula:

aldehyde, isobutylaldehyde, acetone, methylethyl ketone,

diethyl ketone, etc.

Specific starting materials operable in this reductive alkylation process include:

' 7-nitro-6-demethyltetracycline,

7-amino-6-demethyltetracycline, 9-nitro-6-demethyltetracycline, 7,9-dinitro-G-demethyltetracycline, 7,9-diamino-o-demethyltetracycline,

7 -nitro-9-amino-6-demethyltetracycl-ine, 7-amino-9-nitro-6-demethyltetracycline, 7-nitrotetracycline,

9-nitrotetracycline,

7-nitro-5 -l1ydroxytetracycline, 9-nitro-5-hydroxytetracycline, 7-nit-ro-5-hydroxy-6-demethyltetracycline, and 7,9-dinitro-5-hydroxy-6-demethyltetracycline.

The 7- and/or 9-nitro tetracycline starting materials for this process may be prepared in either of two ways.

In the first situation, an appropriate tetracycline may be directly nitrated with nitronium tetrafiuoroborate in a solvent such as nitromethane or .acetonitrile over a period of time of about one hour at room temperature. If a mononitro derivative is desired, then 1 molar equivalent of nitronium tetrafiuoroborate is employed whereas if the dinitro derivative is desired, then 2 molar equivalents of nitronium tetr-afluoroborate are employed. The reaction mixture is then added slowly to a suitable volume of diethyl ether whereupon the desired nitro derivative is precipitated and may be collected by filtration. In the second situation, an appropriate lla-chloro tetracycline is nitrated with nitronium tetr-afluoroborate in a solvent such as nitromethane or acetonitrile over a period of time of about one hour at room temperature. If the mononitro derivative is desired then 1.2 molar equivalents of nitronium tetrafluoroborate are employed whereas if the dinitro derivative is desired, then 2.4 molar equivalents of nitronium tetrafluoroborate are employed. The reaction mixture is then added slowly to a suitable volume of diethyl ether whereupon the desired nitro derivative is precipitated and may be collected by filtration. The

lla-chloro-nitro derivative is then treated with sodium bisulfite in an aqueous solution at a pH of about 6.0 in order to remove the lla-chloro group. In both situations, the separation of isomers (e.g. 7-nitro and 9-nitro deriva tives) may be accomplished by standard crystallization or chromatographic techniques.

The 7- and/or 9-nitro tetracyclines may be reduced either chemically or catalytically to form the corresponding 7- and/ or 9-amino tetracyclines. The catalytic reduction may be carried out in a polar solvent such as Water; a lower alkanol, e.g., methanol, ethanol, etc.; a lower ialkoxy lower alkanol, e.g., Z-methoxyethanol, 2-ethoxyethanol, etc.; or a lower alkanoic acid, e.g., acetic acid, propionic acid, etc.; in a mineral acid solution, e.g., hy-

drochloric acid, sulfuric acid or the like, and in the palladium, rhodium or other metal of the platinum family. A pure metal may be used or the metal amy be used in the form of an oxide or hydroxide and preferably the catalyst is suspended on one of the common carriers such as finely divided alumina, activated charcoal, diatornaceous earth, etc. The reduction may be carried out at temperatures ranging from about C. to about 40 C., and preferably at room temperature, that is around 25 C., and at hydrogen pressures of one to about three atmospheres. The 7- and/ or 9-amino tetracycline so-prepared may be recovered from the reaction mixture by any desired means, as by removal of the catalyst and concentration of the solution. The solution may be evaporated to dryness and purified 7- and/or 9-aminotetracycline may be obtained by precipitation from ethanol-ethyl acetate. The product may be further purified, if desired, by recrystallization from alcohol in a standard manner.

The 7- and/or 9-nitro tetracyclines may also be reduced to 7- and/ or 9-arnino tetracyclines by a chemical reduction process in which the nitro tetracycline is contacted with a hydrogen-producing mixture such as metallic zinc in a mildly acidic medium such as hydrochloric acid, acetic acid, etc. at a temperature of from about 10 C. to about 40 C. and for a time of from about minutes to about two hours. The concentration of the nitro tetracycline in the acidic medium depends upon its solubility. The zinc used for the reaction should preferably be in finely-divided form, for instance, zinc dust, and this material should be used to an extent of about 0.35 part by weight of the metal per part by weight of the nitro tetracycline. Proportions of metal higher than about 5 parts by weight are generally not necessary. The reduced solution contains the desired amino tetracycline which may be recovered therefrom in a standard manner.

The reductive alkylation process may be accomplished by either chemical or catalytic reduction using procedures Well-known to those in the art. Catalytic reduction, which is especially suited for the reductive alkylation of the tetracycline starting compounds set forth above, may be accomplished in a solvent for the tetracycline starting compound in the presence of a carbonyl compound and a metal catalyst and hydrogen gas at pressures from atmospheric to super-atmospheric. Ordinarily, the reductive alkylation is conveniently carried out at hydrogen pressures of from about one to about four atmospheres. Temperature does not appear to be critical in the catalytic hydrogenation. Temperatures of from 0 C. to 50 C., and usually room temperature, are preferred since they generally give best results. The metal catalyst may be of the base metal type, such as nickel or copper chromite, or it may be of the noble metal type, such as finely divided platinum, palladium or rhodium. The noble metal catalysts are advantageously employed on a carrier such as finely divided alumina, activated charcoal, diatomaceous earth, etc. in which form they are commonly available. The hydrogenation is carried out until the desired amount of hydrogen gas is absorbed at which point the hydrogenation is stopped. The solvents selected for the catalytic reduction should be reactioninert, that is, they should not be capable of reaction with the starting materials, product, or hydrogen under the conditions of the reaction. A variety of solvents may be used for this purpose and minimum laboratory experimentation will permit the selection of a suitable solvent for any specific tetracycline starting compound. Generally, the catalytic reductive alkylation may be carried out in solvents such as Water; lower alkanols, e.g. methanol, ethanol; lower alkoxy lower alkanols, e.g. Z-methoxyethanol, 2- ethoxyethanol; tetrahydro-furan; dioxane; dimethylformamide; etc.

The reduction conditions and molar proportions of reactants determine whether a 7- and/or 9-mono(lower alkyl)amino or a 7- and/ or 9-di(lower alkyl)amino derivat'ive is the preponderant product. If the di(lower alkyl)amino derivative is desired than an excess of aldehyde or ketone is employed with a moderate excess of acid and hydrogenation is carried out for an extended period. If the mono(lower alkyl) amino derivative is desired, this may be accomplished by using about one mole of aldehyde or ketone or by terminating the reaction when the theoretical amount of hydrogen has been absorbed. The use of excess acid also has a tendency to produce the mono(lower alkyl)amino derivative rather than the di(lower alkyl)amino derivative even when an excess of aldehyde or ketone is present.

A variety of chemical reducing agents may be used in the reductive alkylation process. These include reduction with active metals in mineral acids, e.g. zinc. tin, or iron in hydrochloric acid; reduction with metal couples such as the copper-zinc couple, the tin-mercury couple, aluminum amalgam, or magnesium amalgam; and reduction with formic acid. Of these, reduction with zinc and hydrochloric acid and reduction with formic acid are preferred. When aqueous system are used in the aforementioned chemical reductive alkylation, it is at times desirable to utilize a water-miscible organic solvent, particularly when the tetracycline starting compound is of limited solubility in the reaction mixture. The watermiscible solvent does not alter the course of the reduction but merely provides for more efficient reduction, e.g. a shorter reaction time by providing more intimate contact of the reagents. A large number of such solvents are available for this purpose and include, among others, dimethylformamide, dimethoxyethane, methanol, ethanol, dioane, tetrahydrofuran, and the like.

The novel products of the present invention are obtained from the reductive alkylation reaction mixtures by standard procedures. For example, the products may be isolated from the catalytic hydrogenation reaction mixtures, after filtration of the catalyst, by precipitation with a solvent such as diethyl ether or hexane or by concentration, usually under reduced pressure, or by a combination of these. Work-up of the chemical reductive alkylation reaction mixtures to obtain the desired products may also be accomplished by known procedures such as precipitation, concentration, solvent extraction, or combinations of these procedures. After isolation, the products may be purified by any of the generally known methods for purification of tetracycline compounds. These include recrystallization from various solvents and mixed solvent systems, chromatographic techniques, and counter current distribution, all of which are usually employed for this purpose.

The novel substituted 7- and/ or 9-amino tetracyclines of the present invention are biologically active and possess the broad spectrum antibacterial activity of the previously known tetracyclines. More particularly, the 7 dimethylamino 6 demethyltetracycline possesses extraordinary activity both orally and parenterally against Staphylococcus aureus, strain Smith, and Staphylococcus acureus, strain Rose, infections in mice.

Staphylococcus aureus, strain Smith, has been studied and described by I. M. Smith, and R. J. Dubos in Journ. Expt. Med., 103, 87 (1956), at the Rockefeller Institute. Staphylococcus wu'reus, strain Smith, is coagulase positive, tellurite negative, and is sensitive to tetracycline, penicillin, streptomycin, erythromycin, carbomycin, neomycin, chloramphenicol and novobiocin in vitro. Attempts have been made for phage typing of this strain, but it has been determined that it is nontypable.

Staphylococcus aureus, strain Rose (ATCC No. 14,154), was isolated clinically from an abscess of a patient who did not respond to treatment with the tetracyclines. This organism has been found to be resistant to the clinically used tetracyclines in vitro and in vivo. Staphylococcus aureus, strain Rose, is coagulase and tellurite positive and is resistant to tetracycline, penicillin, streptomycin, and erythromycin. It is sensitive to carbomycin, meomycin, chloramphenicol and novobiocin in vitro.

Staphylococcus aareus, strain Rose, has been phage-typed with the following results:

Staphylococcus aureus, strain Rose-Phage pattern 80/81.

It has been determined that intravenously against Staphylococcns aureus, strain Rose, infections in mice, 7-dimethylamino-6-demethyltetracycline is about 8 times as potent as tetracycline. When administered in a single oral tubing dose, against Staphylococcus aureus, strain Rose, infection, 7 dimethylamino-6 demethyltetracycline is about 8 times more effective than tetracycline. Additionally, after single oral tubing doses in mice, plasma levels of 7-dimethylamino-6-demethyltetracycline are about 5 times higher than those obtained with demethylchlorotetracycline.

Of special interest is the fact that 7-dimethylamino-6- demethyltetracycline is uniquely active among the tetracyclines in possessing unexpected high oral activity against the tetracycline-resistant strain Staphylococcus aureus, strain Smith. Thus, with oral doses as low as 8 mg./kg., 10 out of 10 mice infected with Staphylococcus aureas, strain Smith, and treated with 7-dimethylamino- 6-demethyltetracycline were alive 5 days after infection, whereas 10 out of 20 of the infected non-treated control mice died within 1 day. Under the same test conditions tetracycline at 64 mg./kg. dose level saved only 7 out of 10 mice infected with Staphylococcus aureus, strain Smith, 5 days after infection.

The new tetracyclines of this invention are amphoteric compounds and hence acid-addition salts, that is both monoand di-salts, may be readily prepared. The pre ferred acids are the non-toxic pharmaceutically acceptable acids, e.g. the mineral acids such as hydrochloric, sulfuric, phosphoric, and the like although organic acids such as tartaric, acetic, and trichloroacetic may also be used. The acid-addition salts may be prepared by treating the new tetracyclines with approximately two equivalents or more of the chosen acid in a suitable solvent.

The invention will be described in greater detail in conjunction with the following specific examples.

EXAMPLE 1 Preparation of nitronium tetrafluoroborate A 17 ml. (20 gm.; specific gravity 1.124) portion of nitromethane and 9.4 ml. (14 gm.) of fuming nitric acid were mixed in a polyethylene container held at 6 C. in a bath consisting of 20% isopropyl alcohol, 80% water and Dry Ice. A 4 ml. (3.94 gm.) portion of hydrofluoric acid was added and the mixture was saturated with boron trifluoride at 6 C. until boron trifluoride fumes were released. The White crystalline precipitate was filtered through a glass funnel under nitrogen atmosphere and then washed with 20 ml. of nitromethane and 20 ml. of l,1,2-t1'ifluorotrichloroethane (Freon 113). The material was transferred to a 3 neck flask and dried in vacuum at 60-70 C. for 1 /2 hours.

EXAMPLE 2 Preparation of 7-nitrotetracycline and 9-nitrotetracycline xg g =272, 445 m with 272/445=2.75

The 7- and 9-nitro isomers were separated as described in Example 5.

6. EXAMPLE 3 Preparation of Ila-blocked tetracycline and nitration to give 7-nitro and 9-nitro-11a-blocked tetracycline A 15 gram portion of tetracycline neutral was dissolved in a solvent mixture of 225 ml. of methanol and 375 ml. of benzene. The solution was evaporated to dryness under reduced pressure. The dried product was then dissolved in 187 ml. of distilled monoglyme. 'To this was added N-chlorosuccinimide and the mixture Was stirred for 3 minutes. The solution was filtered. The clear filtrate was diluted with 200 ml. of water and the resulting suspension was stirred for minutes. The product was iso lated by filtration and dried at 40 C. for about 16 hours yielding 4.8 gm. of pure material having the following spectra:

iggg =268, 340 m with 268/3 l0=4.95

A8 5,? =235, 280, 350 m To a suspension of 1.1 gm. of lla-chlorotetracycline was added 0.388 gm. of nitronium tetrafluoroborate. The resulting solution was stirred for 1 hour at room temperature and then added slowly with stirring to 350 ml. of

ether. The product was filtered, slurry washed with 10 ml. portions of ether and dried at C. and 2.5 mm. of

- pressure for 10 hours. The yield was 1.114 gm. of material having the following spectra:

titg =2e5, 345 m with 265/345=4.3 xgggf =235, 277, 350, 392 m Polarographic analysis have 7-nitro=20.7% and 9-nitro=26.5%.

EXAMPLE 4 Preparation of o-dimethyl-7-nilrotetracycline and 6-demcthyl-9-nitrotctracycline A 1.66 gm. portion of 6-demethyltetracycline was dried from a mixture of 45 ml. of methanol and 75 ml. of benzene. The dried product was then dissolved in ml. of nitromethane. A 0.513 gm. portion of nitronium tetr-afloroborate (NO BF was added to the solution followed by 1 /2 hours of stirring. The solution was then diluted with 1 liter of ether. The light yellow precipitate was filtered and dried at 40 C. for 16 hours yielding 1.383 gm. of crude product having the following spectra:

tggg :265, 354., mg with 265/354=1.48

Aggy MOE :240, 255, 230, 365 m with 380/465 6.3

EXAMPLE 5 Chromatographic separation of the 7-nitro and 9-nitro isomers of 6-demethyltetracycline A gm. portion of acid washed Celite was suspended in a solution of 20 gm. of ethylenediaminetetraacetic acid disodium salt in 500 ml. of water. To this was added 7=nitroz xfig f :260, 352 m with 260/352=1.82

my =235, 280, 375 In with 380/460=3.46

9=nitr-o:

xiii HSO=26O, 361 m with 260/36l=1.6

conversion to 1la-blocked-6-dem'ethyl-7-nitrotetracycline and 11a-bl0cked-6-demethyl-9-nitrotetracycline A 10 gm. portion of 6-demethyltetracycline neutral was dissolved in a solvent mixture of 240 ml. of methanol and 360 ml. of benzene. The solution was evaporated to dryness under reduced pressure. The dried product was dissolved in 400 ml. of distilled dioxane. A solution of 3.22 gm. of N-chlorosuccinimide in 40 ml. of dioxane was added slowly with stirring. The reaction was complete in less than 1 minute. The solution was filtered and the filtrate was diluted with Skellysolve C. The light yellow precipitate was isolated by filtration and dried in a 35 C. vacuum oven for 16 hours. The yield was 10.5 gm. of crude product having the following spectra:

ggllag HzS 4 271 347 m,t with 271/347=5.31

To a suspension of gm. of this 11a-chloro-6-demethyltetracycline in 210 ml. of nitromethane and 1.41 ml. of methanesulfonic acid was added 3.62 gm. of nitronium tetrafluoroborate. The solution was stirred for 2 hours at 2427 C. and then filtered. The filtrate was diluted with 4 liters of ether. The light yellow precipitate was collected and dried in a 35 C. vacuum oven for 16 hours. The yield was 10.5 gm. of crude product having the following spectra:

xii- =257, 347 and 430 m with 257 347=4.e

x333 -275, 390 mp with 380/460 1.46 The 7- and 9-nitro isomers were separated as described in Example 5.

EXAMPLE 7 Reduction of 1 1 tl-chloro-6-demethyl-7 (9) nitrotetracycline to 6-demethyl-7(9)-nitr0tetracycline A 22 gm. portion of a mixture of 11a-chloro-6-demethyl-7-nitrotetracycline and 11a-chloro-6-demethyl=9 nitrotetracycline (prepared as described in Example 6) was suspended in 5 60 ml. of water. The pH was adjusted to 6.0 with 2 N NaOH. To this was added 44 gm. of sodium bisulfite with stirring while the pH was readjusted to 6.0 with 2 N NaOH. Stirring was filtered and the filtrate was acidified to pH 4.0 with concentrated HCl and the brown solid [crop No. 1 (8.8 -gm.)] was isolated by filtration. Acidification was continued. At pH 2.0 a light brown precipitate was obtained [crop No. 2. (3.3 gm.)]. Finally the mother liquor was extracted with butanol at pH 2.0 and the extract was evaporated to dryness under reduced pressure [crop No. 3 (8.6 gm.)]. All three crops were analyzed to be mixtures of salts with the following U.V. spectra:

Crop No. 1:

g t =260, 355, 420 m with 260/355=2.9

waits: mon ggo 260, 372, 430 Inn with 38OI460=L05 Crop No. 2:

i t-.259, 355, 420 my. with 259/355=3.01

y =221, 260, 372 m w th 88 Crop No. 3:

N =261,'352 mi w 6 max 8 EXAMPLE 8 Preparation of 5-hydr0xy-7-nitr0-tetracycline and 5-hydroxy-9-nitr0tetratycline A 1.0 gm. portion of S-hydroxytetracycline, dried from a mixture of 30 ml. of methanol and 30 ml. of benzene as described in Example 3 was dissolved in 40 ml. of nitromethane. A 0.3 gm. portion of nitronium tetrafluoroborate was added and the solution was stirred for 4 hours at 25 C. The solution was diluted with 500 ml. of ether and the product was isolated by filtration. The yield after drying at 35 C. was 1.706 grn. having the following spectra:

255, 350, 415 m with 250 350=4.05

h9g5 =253, 275, 400 mg with 3s0/460=1.94

EXAMPLE 9 Preparation of 11a blocked 5 hydroxytetracycline and conversion to Ho blocked 5 hydroxy 7 nitrotetracycline and 11a blocked 5 hydroxy 9 nitrotetracycline A 3.0 gm. portion of S-hydroxytetracycline neutral was dissolved in a solvent mixture consisting of ml. of methanol and 225 ml. of benzene. The solution was evaporated to dryness under reduced pressure. The dried product was dissolved in 135 ml. of distilled dioxane. A 1.05 gm. portion of N-chlorosuccinimide in 15 ml. of dioxane was added slowly with stirring. The reaction was complete in 3 minutes. The solution was filtered. The filtrate was diluted with 390 ml. of Skellysolve C. The product was isolated by filtration and dried at 40 C. for 15 hours. The yield was 2.7 gm.

for 10 hours. The yield was 0.941 gm. having the following spectra:

r g-g g =258, 345 In with 25s/345=4.62 Nth? """=275, 400 mp.

Polarographic analysis showed 21.6% 7-nitro isomer and 50.9% 9-nitro isomer.

EXAMPLE 10 Reductive alkylation of 6-dem'ethyl-7-nitr0tetraoycline to give 6-demethyl-7-dimethylam-inotetracycline A 5 milligram portion of 6-demthyl-7-nitrotetracyc1ine was dissolved in 1 ml. of methanol containing 0.3 ml. of 0.2 N HCl. To this was added 5 mg. of 10% palladium on carbon and 0.025 ml. of 40% formaldehyde. The mixture was reduced catalytically under atmospheric pressure for 1 hour and 30 minutes at room temperature. The catalyst was filtered ofi and washed with 1 m1. of methanol. The filtrate and wash was evaporated under reduced pressure to dryness. Chromatographic analysis showed the presence of 6-demethyl-7-dimethylaminotetracycline.

A turbidimetric assay of 6-demethyl-7-nitrotetracycline gave 585 mcg./mg. as tetracycline. The same assay of 6- demethyl-7-dimethylaminotetracycline gave 986 meg/mg. as tetracycline.

xgtg -266, 354 m O.D. Ratio 266/354=2.48 k951i =245, 265, 385 my O.D. Ratio 265/385=1.'Z,

9 EXAMPLE 11 A solution of 240 mg. of 6-demethyl-7-nitr0tetracycline free base, 50 ml. of methanol, 1.22 ml. of 40% aqueous formaldehyde and 0.124 ml. of cone. HCl was mixed with 120 mg. of 10% palladium-on-ca-rbon catalyst. The mixture was reduced at atmospheric pressure and room temperature for one and a half hours. The hydrogen uptake could not be measured accurately because of the vapors of methanol which caused a negative reading. The catalyst was then filtered and the filtrate evaporated to dryness. This material was chromatographed on 55 g. of acidwashed Celite at pH 6.6 using the system heptane:ethyl acetatetrnethanoltwater (60:40:15:6). An essentially pure material was obtained (54.4 mg.) which analyzed as follows:

x353 =264, 354 mg with R 264/354=1.59 x15. =245, 270 (shoulder), 384 m Paper chromatography in a system nitromethane: benzenezpyridinezpH 3.4 phosphate/citrate bufier (20: 10:3:3) run at C. showed spots at R =0.7 and 0.03 (faint) after treatment with magnesium acetate solution. In the system BuOH, ammonia, pH 8.0 showed two spots at R =0.5 and 0.26 after treatment with the magnesium acetate solution. Turbidimetric assay against St. aureus was 1700'y/mg. as tetracycline.

EXAMPLE 12 Reductive ethylation of 7-nitrotetracycline to yield 7-diethylaminotetracycline EXAMPLE 13 Preparation of 7-dimethylamina-S-hydroxytetracycline A solution of 1.0 g. of 7-nitro-S-hydroxytetracycline in 75 ml. of methanol, 6 ml. of 40% aqueous formaldehyde and 0.52 ml. of 35% aqueous hydrochloric acid was catalytically reduced in the presence of 250 mg. of palladium on charcoal catalyst until five moles of hydrogen were absorbed. The catalyst was filtered off and the filtrate evaporated to dryness. When purified by partition column chromatography, there was obtained 620 mg. of 7-dimethylamino-S-hydroxytetracycline. Paper chromatography in a system containing nitromethane, benzene, pyridine and pH 3.4 phosphate-citrate buifer run at 0 C. showed the product as the major spot.

dried residue after evaporation as the methanol was chromatographed to separate the isomeric 7- and 9-dimethylaminotetracyclines. The chromatographic system used consisted of a support of 350 g. of acid washed Celite 545 (a commercial diatomaceous earth) and the solvent system heptanezethyl acetatezmethanolzwater (60:40:1516 by volume). The solvent fractions containing the 7-dimethylarninotet-racycline and 9 dimethylaminotetracycline were evaporated to dryness. The residues were dissolved in warm chloroform, filtered to remove traces of buffer from the column and evaporated to dryness to recover 7-dimethylaminotetracycline and 9-dirnethylaminotetracycline.

What is claimed is:

1. A compound selected from the group consisting of tetracyclines of the formula:

OHO OH wherein R is selected from the group consisting of hydrogen and hydroxy, R is selected from the group consisting of hydrogen and methyl, and R and R are each selected from the group consisting of hydrogen, mono (lower alkyl)amino and di(lower alkyl)amino With the proviso that R and R cannot both be hydrogen; and the nontoxic pharmaceutically acceptable acid-addition salts thereof.

2. A compound according to claim 1 wherein R is hydrogen, R is hydrogen, R is dimethylamino and R i hydrogen.

3. A compound according to claim 1 wherein R is hydrogen, R is hydrogen, R is hydrogen and R is methylamino.

4. A compound according to claim 1 wherein R is hydrogen, R is hydrogen, R is diethylamino and R is hydrogen.

5. A compound according to claim 1 wherein R is hydrogen, R is hydrogen, R is dimethylamino and R is hydrogen; as the hydrochloric acid salt.

6. A compound according to claim 1 wherein R is hydrogen, R is hydrogen, R is dimethylamino and R i hydrogen; as the sulfuric acid salt.

7. A compound according to claim 1 wherein R is hydrogen, R is methyl, R is dimethylamino and R is hydrogen.

8. A compound according to claim 1 wherein R is hydroxy, R is methyl, R is dimethylamino and R is hydrogen.

9. A compound according to claim 1 wherein R is hydroxy, R is hydrogen, R is dimethylamino and R is hydrogen.

10. A compound according to claim 1 wherein R is hydrogen, R is methyl, R is dimethylamino and R is hydrogen; as the hydrochloric acid salt.

References Cited UNITED STATES PATENTS 3,165,551 l/l965 Blackwood et a1. 260559 NICHOLAS s. RIZZO', Primary Examiner. 

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF TETRACYCLINES OF THE FORMULA: 